Fungal infections are becoming an increasingly significant cause of morbidity and mortality. In the course of the 1980s, the rate of bloodstream infection by Candida albicans surged by 48%. Patients at particular risk of mycoses are those with diminished immune defenses—not only organ transplant patients or those receiving intensive treatment for cancer, but also diabetics or people with indwelling catheters.
Fungal infections account for a large number of AIDS-defining diagnoses and complicate the course of most patients with HIV disease. The ECHO estimates that there will be more than 20 million HIV-infected adults alive in the year 2000. The impact of fungi disease in AIDS patients is immense because of their recurring nature. Of the 25 conditions which make up the case definition for AIDS, seven are caused by fungal pathogens. Of the most frequent AIDS indicator diseases occurring among homosexual/bisexual men, 70% were fungal infections, with Pneumocystis carinii pneumonia (PCP) being the most common of all diseases.
Fungi occur in a wide variety of forms, from yeasts (single-celled organisms which reproduce by budding) and moulds (which occur in long filaments known as hyphae) to the dimorphic fungi which have a chameleon-like ability to behave as yeasts in one environment and mould in another.
Of the many different types of fungi only a few have the potential to cause disease, and the severity of their effects varies widely. Superficial mycoses, caused by a fungus such as Epidermophyton, Microsporum, Trichophyton or Sporothrix growing on the body surface (skin, nails or hair) are unpleasant but usually mild infections.
Deep mycoses, involving the internal organs, are often life-threatening. The fungi Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Histoplasma are responsible for deep mycoses, and pose tremendous challenges for clinicians. Clinically, candidiasis and aspergillosis account for between 80% and 90% of systemic fungal infections in immunocompromised patients. Recently, infections caused by Pneumocystis carinii are more frequently found in AIDS patients.
Pneumocystis carinii is a major opportunistic infectious agent in immunocompromised patients, causing pneumonia which has a high mortality rate if the patient is not properly treated (Stringer J. R., 1996). Therefore, timely diagnosis of P. carinii pneumonia (PCP) is critical for patient management. Currently, diagnosis of PCP is usually made by morphological demonstration of organisms in bronchoalveolar lavage (BAL) fluid, induced sputum, or specimens obtained by open lung biopsy (Cregan et al., 1990). Although morphological diagnosis is rapid, it requires highly experienced personnel and good specimens. Given the inability to culture P. carinii in vitro, molecular biology-based methods have been used for the detection of this pathogen and to study P. carinii epidemiology (Lu et al., 1995).
Candidoses comprise a range of human opportunistic infections which may occur in either acute or chronic forms. Candida infections frequently arise on the mucosal surfaces of the mouth or vagina. Chronic hyperplastic candidosis of the oral mucosa is of particular importance since it has been associated with the development of squamous cell carcinoma. In addition to superficial lesions, deeper candida infections, such as esophagitis and endocarditis, may occur, particularly in immunocompromised individuals (Heimdahl et al., 1990).
In the past, many studies of candidosis have not identified candidal isolates to species level. Indeed, in the case of chronic hyperplastic candidosis, reporting is usually limited to the presence of structures consistent with candidal hyphae following histological examination of lesional tissue. However, it is becoming increasingly recognized that both species and subspecies of Candida differ in their ability to cause disease. Traditional methods used for the identification and typing of clinical isolates of Candida include morphological and biochemical analysis (Williamson et al., 1986), colony morphotyping (Soll, D. R. 1992), resistogram typing (Sobczak, H., 1985), and serotyping (Brawner, D. L., 1991). These techniques are time-consuming, and their reliance on phenotypic expression makes them potentially unreliable. An alternative method would be one based on genotypic properties. Genotypic methods have been used for the detection and typing of Candida strains (Bart-Delabesse et al., 1993; Holmes et al., 1994), but have been used less frequently for differentiation of species.
Other medically important Candida species, next to the most frequently isolated pathogen C. albicans, are C. glabrata. C. krusei and C. tropicalis. The latter species are much less susceptible to classical antifungal drugs.
The genus Cryptococcus contains many species, wherein Cryptococcus neoformans is considered the only human pathogen. Initial cryptococcal infection begins by inhalation of the fungus into the lungs, usually followed by hematogeneous spread to the brain and meninges. Involvement of the skin, bores, and joints is seen, and Cryptococcus neoformans is often cultured from the urine of patients with disseminated infection. In patients without HIV infection, cryptococcosis, particularly cryptococcal meningitis, usually is seen in association with underlying conditions such as lupus erythematosis, sarcoidosis, leukemia, lymphomas, and Cushing's syndrome (Chuck et al., 1989).
Cryptococcosis is one of the defining diseases associated with AIDS. Patients with cryptococcosis and serologic evidence of HIV infections are considered to have AIDS. In nearly 45% of AIDS patients, cryptococcosis was reported as the first AIDS-defining illness. Because none of the representing signs or symptoms of cryptococcal meningitis (such as headache, fever, and malaise) are sufficiently characteristic to distinguish it from other infections that occur in patients with AIDS, determining cryptococcal antigen titers and culturing blood and cerebrospinal fluid are useful in making a diagnosis (Chuck et al. 1989).
The clinical diagnosis of pulmonary cryptococcosis in patients without underlying diseases is generally difficult. Since the diagnosis is often established only by examination of tissue obtained from lung biopsy specimens, other more sensitive and specific methods are needed for the simple and fast detection of the fungus. One such approach involves the detection of fungal antigens in serum (Kohno et al., 1993). However, evaluations of serological assays for detecting cryptococcal antigen showed false-positive reactions with sera from patients infected with Trichosporon beigelii (Kohno et al., 1993).
Aspergillosis is now considered the second most common fungal infection requiring hospitalization. In patients with positive fungus cultures, Aspergillus species are the second most common isolate after Candida species (Goodwin et al., 1992). The pathological responses caused by members of the genus Aspergillus vary in severity and clinical course and may occur as both primary and secondary infections (Rinaldi, M. G., 1988).
Invasive aspergillosis (IA) is a life-threatening condition in immunocompromised patients, particularly those treated with chemotherapy for hematologic malignancies or those receiving high-dose corticosteroids (Fisher et al., 1981). An early diagnosis of aspergillosis is of great importance because early treatment may resolve this potentially fatal infection. Unfortunately, the diagnosis of IA remains difficult and sometimes is confirmed only at autopsy. At present, a firm diagnosis is established by histological examination of tissue samples obtained during open lung biopsy as well as by detecting the causative pathogenic fungi in clinical samples. Serological tests such as those involving the detection of antibodies for Aspergillus species are less helpful because of the poor antibody responses in immunocompromised patients. In addition, the methods used for detecting circulating Aspergillus antigens, such as radioimmunoassay, immunoblotting assay, enzyme immunoassay, and the latex agglutination test, have poor sensitivity (Rogers et al., 1990; Sabetta et al., 1985).
Recently, for the diagnosis of IA, PCR has been used to detect DNA specific for Aspergillus species in bronchoalveolar lavage (BAL) fluid from patients with IA (Bretage et al., 1995).
Laboratory diagnosis of fungal infections is often problematic. Fungi are often difficult to culture from readily accessible samples, such as patient's urine, blood or sputum. And because fungi are ubiquitous in nature, a single positive culture from urine or sputum is of limited clinical value. The possibility of contamination is always a very real consideration when interpreting laboratory results. A finding of Aspergillus in sputum, for example, is in isolation of limited value and must be evaluated in the context of the patient's clinical signs and symptoms. Often tissue biopsies (from lung or brain) are needed for definitive diagnosis and blood cultures should be carried out for all patients. Isolation of yeast—such as Candida—from blood is highly predictive of invasive fungal disease. However Candida is cultured from blood in less than 20% of patients with disseminated candidiasis.
Because of the limitation of culture techniques many researchers have tried to find specific antibodies against Candida and Aspergillus in sequence by using the titre of antibodies as diagnostic criterium. However, the sensitivity of these assays is very low—often less than 50%—as many immunocompromised patients have difficulty raising an adequate immune response. Because of its poor sensitivity, immunodiagnosis of fungal infection is not cost-effective. It often gives a false negative result, and may also lead to fungal infections being diagnosed where none exist, leading to the inappropriate administration of antifungal drugs.
Because of the shortcomings of antibody detection, much attention has been directed to tests which detect fungal antigens or metabolites in body fluids. A major problem is the transient nature of antigens in serum. For most antigen detection tests the overall sensitivity is unacceptably low, although multiple serum sampling somewhat improves detection of antigenaemia.
Moreover, none of the above-cited methods allows, the identification of the fungus up to the species level. Efficient treatment regimens of fungal diseases require a correct identification of the fungus at the species level. For example, certain Candida species such as C. glabrata and C. krusei are less susceptible to the classical azole drugs.
The diagnostics of mycoses is an area where there is a great need for new sophisticated techniques. As already seen in virology and to some degree in bacteriology, the use of specific DNA probes, accompanied by DNA or RNA amplification systems, for the diagnosis of fungal infection may prove useful, and may revolutionize laboratory diagnosis and management of patients with serious fungal disease.
Recently, several methods for detection of fungal pathogens using DNA technology have been described. Genes encoding the rRNA, especially the 18S and 28S rRNA genes, have been frequently used as a target for developing species specific probes (e.g. U.S. Pat. No. 5,827,651; Einsele et al. 1997). Others report on the use of the Internal Transcribed Spacer (ITS) region, located in between the 18S and 28S rRNA genes, as a target region for the specific detection of fungi. Lu et al. (1995) describe the subtyping of Pneumocystis carinii strains using probes originating from the ITS region. Williams et al. (1995) demonstrate identification of Candida species by PCR amplification and restriction length polymorphism analysis of the ITS amplified regions. Kumeda and Asao (1996) use PCR amplification and single strand conformation polymorphism (sscp) analysis of the ITS region to differentiate species of Aspergillus. U.S. Pat. No. 5,693,501 describes specific primers originating from the ITS-1 region for detection of Histoplasma capsulatum. A number of patent applications (WO98/50584; WO95/29260; U.S. Pat. No. 5,814,463; U.S. Pat. No. 5,955,274) describe detection and differentiation of different plant fungal pathogens based on specific amplification of or hybridization to ITS-region sequences.
Detection of Candida species based on ITS-2 region sequences has been described by several groups. Fujita et al. (1995) describe ITS-2 probes for different Candida species and methods for detection and differentiation after a general amplification step with universal primers ITS3 and ITS4. Elie et al. (1998) and several related patent applications (WO98/11257; WO99/06596; U.S. Pat. No. 5,426,027) describe a set of 18 Candida species probes originating from the ITS-2 region. Shin et al. (1999) describe detection and differentation of three Candida species in a single reaction tube, using amplification with the universal primers ITS3 and ITS4 and hybridization to ITS-2 probes. Botelho and Planta (1994) describe probes for Candida albicans, derived from the ITS-1 or ITS-2 regions. The ITS-2 region probes show a better species specificity.
Species specific probes originating from the ITS-region of other medically important fungal species, such as species belonging to Aspergillus, Cryptococcus, or Pneumocystis have not been described yet. Moreover, methods for simultaneous detection and differentiation of a wide variety of fungal species with clinical importance have not been described yet. Such methods would provide an answer to the need for rapid, highly sensitive and species specific detection of fungal pathogens in clinical samples, allowing a quick installment of efficient treatment regimens, and close monitoring of a patient's progression.